Mammalian hematopoietic (blood) cells provide a diverse range of physiologic activities. Hematopoietic cells are divided into lymphoid, myeloid and erythroid lineages. The lymphoid lineage, comprising B, T and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes, as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers.
All publications cited herein are hereby incorporated herein by reference in their entirety.
Despite the diversity of the nature, morphology, characteristics and function of hematopoietic cells, it is presently believed that these cells are derived from a single cell population, termed hematopoietic "stem cells." Unlike more "mature" blood cells, stem cells are capable of self-regeneration but may also divide into progenitor cells that are no longer pluripotent and have a limited self-regeneration. These progenitor cells divide repeatedly to form more mature cells which eventually become terminally differentiated to form the various mature hematopoietic cells. Thus the large number of mature hematopoietic cells is derived from a small reservoir of stem cells by a process of proliferation and differentiation.
Progenitor cells mature into bipotential cells and then become lineage committed, that is, are incapable of maturing into more than one lineage. The use of the words progenitor or progenitor cells indicates cell populations which are no longer stem cells but which have not yet become terminally differentiated. The use of the word lymphoid, myeloid or erythroid in conjunction with progenitor indicates the potential cell populations into which the progenitor is capable of maturing.
Highly purified populations of stem cells currently find use in repopulation of the entire hematopoietic system. Purified progenitor cells of individual lineages would find use in repopulating or augmenting the various lineages. As progenitors are not believed to be extensively self-regenerating, the repopulation or augmentation would be limited.
Stem cells and progenitor cells constitute only a small percentage of the total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence of a variety of cell surface protein "markers." Such markers may be either specific to a particular lineage or be present on more than one cell type. The markers also change with stages of differentiation. Currently, it is not known how many of the markers associated with differentiated cells are also present on stem and progenitor cells. One marker, CD34, is found on stem cells and a significant number of progenitors. U.S. Pat. No. 5,061,620 describes a composition comprising human stem cells.
Table 1 summarizes probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood. As used herein both infra, supra and in Table 1, the negative sign or, uppercase negative sign, (.sup.-) means that the level of the specified marker is undetectable above Ig isotype controls by immunoflow cytometry analysis, and may include cells with very low expression of the specified marker that would be below the sensitivity threshold of the technique.
TABLE 1 __________________________________________________________________________ Probable Stem Cell Phenotypes Natural Killer and B cell Myelomonocytic Other T cell markers markers Markers Markers P-gp CD2 CD3 CD8 CD10 CD19 CD20 CD14 CD15 CD16 CD33 CD34 CD38 HLA-DR C-Kit Thy Rho Activity __________________________________________________________________________ Fetal - - - - - - - - - ? + - + + + lo + Bone Marrow Adult - - - - - - - - - - + ? lo/- + + lo + Bone Marrow Adult - - - - - - - - - lo/- + ? lo/- ? + lo + Mobilized Peripheral Blood __________________________________________________________________________
The exact series of differentiation steps which occurs from stem cells to lineage commitment and to terminal differentiation is unknown; likewise, the various subpopulations of cells involved have not been well characterized.
Lymphocytes are highly specialized hematopoietic cells. During the development of the B and T lymphoid lineages, phenotypic and molecular differentiation of primitive cells leads to mature stages where rearrangement of the lymphocyte antigen receptors occur, namely the immunoglobulin (Ig) or T-cell receptor (TCR) chains. Van Noesel and Lier (1993) Blood 82:363-373; and Godfrey and Zlotnik (1993) Immunol. Today 14:547-553. Commitment to the B-cell lineage, expression of the B-cell receptor complex and Ig gene rearrangements take place in the bone marrow or fetal liver. Uckun (1990) Blood 76:1908-1923; and Li et al. (1993) J. Exp. Med. 178:951-960.
In man, the extensive analyses of leukemias, fetal liver and bone marrow have shown that the earliest recognizable population of cells committed to the B lineage express the markers CD34, HLA-DR and CD10 and have intranuclear terminal deoxynucleotidyl transferase activity with Ig genes in germline configuration. Uckun (1990). The marker CD19 is subsequently expressed and remains expressed throughout most later stages of B-cell differentiation, which stages are further identified by the expression of an array of markers including surface Ig. The marker CD2 is transiently found on early B-cell precursors at the time of CD19 expression, so the earliest B-cell precursor can be identified by expression of CD34 and CD10 but in the absence of CD19 and CD2. Uckun (1990). CD10, or CALLA, is a neutral endopeptidase expressed by several hematopoietic and non-hematopoietic cells. LeBien and McCormack (1989) Blood 73:625.
Unlike B-cell differentiation, T-cell development requires passage of T-progenitor cells through the thymus gland to achieve efficient: T-cell receptor (TCR) rearrangement and major histocompatibility complex (MHC)-restriction. At the thymic stage, immature T cells are called thymocytes. The intrathymic stages of T-cell development have been extensively studied in mice and to a lesser extent in man. Godfrey and Zlotnik (1993); Galy et al. (1993) J. Exp. Med. 178:391-401; Terstappen et al. (1992) Blood 79:666-677; and Sanchez et al. (1993) J. Exp. Med. 178:1857-1866. With use of T-cell differentiation assays and multiparameter flow cytometry, it has been shown that CD34 is expressed on the most immature thymocytes which lack cell surface expression of CD1, CD4, CD8 and CD3 antigens. Galy et al. (1993); and Terstappen et al. (1992). CD34 levels decrease while T-cell maturation proceeds, as is the case with maturation along the myeloid, erythroid and B-cell lineages. Terstappen et al. (1991) Blood 77:1218-1227. Studies in animals using mice or quail/chick chimeras and studies in man with constructs of fetal liver and thymus implanted into surrogate severe combined immunodeficiency (SCID) mice, have shown that a constant input of hematopoietic cells is needed to sustain thymopoiesis. Le Douarin and Jotereau (1973) Nature New Biol. 246:25-27; Scollay et al. (1986) Immunol. Rev. 91:129-157; and McCune et al. (1988) Science 241:1632-1639.
The nature of the pro-thymocyte progenitor is however ill-defined. In man, pre-thymic cells with T-cell differentiative capability have been retrieved from various hematopoietic tissues. Intrathymic T-cell reconstitution is achieved following injection of CD34.sup.+ cells devoid of lineage-specific antigens (Lin.sup.-) isolated from fetal liver (FL). Galy et al. (1993); and Peault et al. (1991) J. Exp. Med. 174:1233-1286. Further fractionation of the CD34.sup.+ Lin.sup.- FL cells has shown that T-lymphoid potential is present in both the CD7.sup.- and CD7dull subsets. Barcena et al. (1393) Blood 82:3401-3414. T-cell differentiation can be initiated with CD34.sup.+ Lin.sup.- fetal bone marrow (FBM) cells and is found in both the CD34.sup.+ Thy-1.sup.+ and CD34.sup.+ Thy-1.sup.- compartments. Baum et al. (1992) Proc. Natl. Acad. Sci. USA 89:2802-2804.
Differentiation into thymocytes can also be achieved with adult tissues. CD34.sup.+ Lin.sup.- cells isolated from normal adult bone marrow (ABM) or from the apheresed cytokine-mobilized peripheral blood (MPB) of cancer patients have recently been shown to undergo de novo thymopoiesis and subsequently proceed toward complete maturation into TCR.alpha..beta..sup.+ and TCR.gamma..delta..sup.+ T-cells. Galy et al. (1994) Blood 84:104-110.
It has been recently reported that after in utero injection into sheep fetuses, CD34.sup.+ HLA.sup.- DR.sup.- cells isolated from human ABM provide long-term (7 months) multilineage hematopoiesis, including production of mature T-cells and B-cells. Srour et al. (1993) Blood 82:3333-3342. These reports provide a preliminary mapping of pre-thymic T-progenitor cell activity, but the phenotypic composition of the T progenitor cell pool as well as the hierarchical ordering of its components remain largely unexplored.
In contrast, extensive phenotypic fractionation of ABM CD34.sup.+ cells has been performed to study myelopoiesis and erythropoiesis. Terstappen et al. (1991); Baum et al. (1992); Lansdorp and Dragowska (1992) J. Exp. Med. 175:1501-1509; Srour et al. (1991) Blood Cells 17:287-295; Craig et al. (1993) J. Exp. Med. 177:1331-1342; and Udomsakdi et al. (1991) Exp. Hematol. 19:338-342. In particular, CD45 isoforms have been useful markers to distinguish primitive from committed myeloid cells. Lansdorp et al. (1990) J. Exp. Med. 172:363-366. CD45 antigens are protein tyrosine phosphatases which exist in various isoforms created by alternative splicing. Thomas (1989) Ann. Rev. Immunol. 7:339-369. The high molecular isoforms (p205-p220) have been designated CD45RA and are not expressed on the cell surface of ABM primitive progenitors having long-term culture initiating-cell (LTCIC) activity. Lansdorp and Dragowska (1992). In contrast, CD45RA is found on less primitive bone marrow progenitors as well as on subsets of mature T-cells where it is thought to correlate with specific functional properties (naive T-cells). Sanders et al. (1988) Immunol. Today 9:195-199.
CD45RA is also expressed on a fraction of thymocytes, particularly on cells at a very early stage of intrathymic development. Deans et al. (1991) J. Immunol. 147:4060-4068. It has actually been speculated with the use of multiparameter flow cytometry that the immediate pre-thymic bone marrow-derived progenitor cells might express CD45RA, but functional evidence has not been provided. Terstappen et al. (1992).
The definitive T cell marker is the T cell antigen receptor (TCR). There are presently two defined types of TCR; TCR-2 is a heterodimer of 2 disulfide-linked transmembrane polypeptides (.alpha. and .beta.), TCR-1 is structurally similar but consists of .gamma. and .delta. polypeptides. The .alpha. and .beta. or .gamma. and .delta. polypeptides form a heterodimer which contains an antigen recognition site. These heterodimers recognize antigen in association with MHC molecules on the surface of antigen-presenting cells. All of these proteins contain a variable region which contributes to the antigen recognition site and a constant region which forms the bulk of the molecule and includes the transmembrane region and cytoplasmic tail. Both receptors are associated with a complex of polypeptides making up the CD3 complex. The CD3 complex comprises the .gamma., .zeta. and .epsilon. transmembrane polypeptides. The CD3 complex mediates signal transduction when T cells are activated by antigen binding to the TCR.
Approximately 95% of blood T cells express TCR-2 and up to 5% have TCR-1. The TCR-2 bearing cells can be subdivided further into two distinct non-overlapping populations. CD4.sup.+ T cells which generally recognize antigens in association with MHC class II molecules, and CD8.sup.+ T cells which recognize antigens in association with MHC class I molecules.
A number of markers are carried by B cells but not by resting T cells. The majority of B cells carry MHC class II antigens which are important in cooperation with T cells. Fc receptors for IgG (FcRII, CDw32) are also present.